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  • C terminus of nucleotide binding domain 1 contains critical features for cystic fibrosis transmembrane conductance regulator trafficking and activation. 20435887

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest beta strands (beta(c)5 and beta(c)6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the beta(c)5-beta(c)6 hairpin, within the beta(c)5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the beta(c)6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3480
    Nombre del producto:
    Anti-CFTR Antibody, a.a. 1370-1380, clone M3A7

  • STE20/SPS1-related proline/alanine-rich kinase is involved in plasticity of GABA signaling function in a mouse model of acquired epilepsy. 24058604

    The intracellular concentration of chloride ([Cl(-)]i) determines the strength and polarity of GABA neurotransmission. STE20/SPS1-related proline/alanine-rich kinase (SPAK) is known as an indirect regulator of [Cl(-)]i for its activation of Na-K-2 Cl(-)co-transporters (NKCC) and inhibition of K-Cl(-)co-transporters (KCC) in many organs. NKCC1 or KCC2 expression changes have been demonstrated previously in the hippocampal neurons of mice with pilocarpine-induced status epilepticus (PISE). However, it remains unclear whether SPAK modulates [Cl(-)]i via NKCC1 or KCC2 in the brain. Also, there are no data clearly characterizing SPAK expression in cortical or hippocampal neurons or confirming an association between SPAK and epilepsy. In the present study, we examined SPAK expression and co-expression with NKCC1 and KCC2 in the hippocampal neurons of mice with PISE, and we investigated alterations in SPAK expression in the hippocampus of such mice. Significant increases in SPAK mRNA and protein levels were detected during various stages of PISE in the PISE mice in comparison to levels in age-matched sham (control) and blank treatment (control) mice. SPAK and NKCC1 expression increased in vitro, while KCC2 was down-regulated in hippocampal neurons following hypoxic conditioning. However, SPAK overexpression did not influence the expression levels of NKCC1 or KCC2. Using co-immunoprecipitation, we determined that the intensity of interaction between SPAK and NKCC1 and between SPAK and KCC2 increased markedly after oxygen-deprivation, whereas SPAK overexpression strengthened the relationships. The [Cl(-)]i of hippocampal neurons changed in a corresponding manner under the different conditions. Our data suggests that SPAK is involved in the plasticity of GABA signaling function in acquired epilepsy via adjustment of [Cl(-)]i in hippocampal neurons.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation. 10085301

    Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, beta cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized beta cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1675
    Nombre del producto:
    Anti-Cytokeratin 19 Antibody, clone E6

  • EGFR kinase regulates volume-sensitive chloride current elicited by integrin stretch via PI-3K and NADPH oxidase in ventricular myocytes. 16505146

    Stretch of beta1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl(-) SAC resembles the volume-sensitive Cl(-) current (I(Cl,swell)). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl(-) SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for beta1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl(-) SAC that was 1.13 +/- 0.10 pA/pF at +40 mV. AG1478 (10 muM), an EGFR kinase blocker, inhibited 93 +/- 13% of Cl(-) SAC, and intracellular pretreatment with 1 muM AG1478 markedly suppressed Cl(-) SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl(-) current (0.81 +/- 0.05 pA/pF at +40 mV) that was fully blocked by 10 muM tamoxifen, an I(Cl,swell) blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 microM), blockers of PI-3K, inhibited Cl(-) SAC by 67 +/- 6% and 91 +/- 25% respectively, and the EGF-induced Cl(-) current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl(-) current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl(-) SAC and EGF-induced Cl(-) current by 88 +/- 9% and 127 +/- 11%, respectively. These results suggest that beta1 integrin stretch activates Cl(-) SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl(-) SAC and the EGF-induced Cl(-) currents are likely to be the volume-sensitive Cl(-) current, I(Cl,swell).
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2250
    Nombre del producto:
    Anti-Integrin β1 Antibody, clone 21C8

  • Carbonic anhydrase XIV in the normal and hypertrophic myocardium. 22227327

    Two AE3 transcripts, full-length (AE3fl) and cardiac (AE3c) are expressed in the heart. AE3 catalyzes electroneutral Cl(-)/HCO(3)(-) exchange across cardiomyocyte sarcolemma. AE proteins associate with carbonic anhydrases (CA), including CAII and CAIV, forming a HCO(3)(-) transport metabolon (BTM), increasing HCO(3)(-) fluxes and regulating cardiomyocytes pH. CAXIV, which is also expressed in the heart's sarcolemma, is a transmembrane enzyme with an extracellular catalytic domain. Herein, AE3/CAXIV physical association was examined by coimmunoprecipitation using rodent heart lysates. CAXIV immunoprecipitated with anti-AE3 antibody and both AE3fl and AE3c were reciprocally immunoprecipitated using anti-CAXIV antibody, indicating AE3fl-AE3c/CAXIV interaction in the myocardium. Coimmunoprecipitation experiments on heart lysates from a mouse with targeted disruption of the ae3 gene, failed to pull down AE3 with the CAXIV antibody. Confocal images demonstrated colocalization of CAXIV and AE3 in mouse ventricular myocytes. Functional association of AE3fl and CAXIV was examined in isolated hypertrophic rat cardiomyocytes, using fluorescence measurements of BCECF to monitor cytosolic pH. Hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHR) presented elevated myocardial AE-mediated Cl(-)/HCO(3)(-) exchange activity (J(HCO3-) mM.min(-1)) compared to normal (Wistar) rats (7.5±1.3, n=4 versus 2.9±0.1, n=6, respectively). AE3fl, AE3c, CAII, CAIV, and CAIX protein expressions were similar in SHR and Wistar rat hearts. However, immunoblots revealed a twofold increase of CAXIV protein expression in the SHR myocardium compared to normal hearts (n=11). Furthermore, the CA-inhibitor, benzolamide, neutralized the stimulatory effect of extracellular CA on AE3 transport activity (3.7±1.5, n=3), normalizing AE3-dependent HCO(3)(-) fluxes in SHR. CAXIV/AE3 interaction constitutes an extracellular component of a BTM which potentiates AE3-mediated HCO(3)(-) transport in the heart. Increased CAXIV expression and consequent AE3/CAXIV complex formation would render AE3 hyperactive in the SHR heart.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB374
    Nombre del producto:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Expression of TMEM16A and SLC4A4 in human pancreatic islets. 22415075

    Stimulation of insulin release by D-glucose is accompanied by Cl(-) and HCO(3)(-) efflux from pancreatic islet cells. The efflux of these anions may involve volume-regulated anion channels, including possibly TMEM16A, and the Na(+)-HCO(3)(-)-cotransporter SLC4A4. The present study was designed to explore the expression of both TMEM16A and SLC4A4 in human pancreatic islets.Pancreases were obtained from human cadaveric donors. Immunodetection of TMEM16A and SLC4A4 was performed by immunohistochemistry on sections of fixed pancreas, while real-time PCR for the study of corresponding gene expression was performed on RNA extracted from both total pancreatic pieces and isolated pancreatic islets.RT-PCR yielded lower levels of SLC4A4 in isolated islets than in the total pancreas, whilst a mirror image prevailed for TMEM16A mRNA. Immunohistochemistry of human pancreas, however, indicated comparable immunostaining of SLC4A4 in insulin-producing cells and exocrine pancreatic cells, whilst that of TMEM16A appeared less pronounced in insulin-producing cells than in exocrine cells.The present findings support the view that, in humans like in rodent, the regulation of anion fluxes in insulin-producing cells may involve both SLC4A4 and TMEM16A.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB3212

  • Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment. 11526230

    There is evidence both for and against Na(+)- and Cl(-)-dependent neurotransmitter transporters forming oligomers. We found that cross-linking the human dopamine transporter (DAT), which is heterologously expressed in human embryonic kidney 293 cells, either with copper phenanthroline (CuP) or the bifunctional reagent bis-(2-methanethiosulfonatoethyl)amine hydrochloride (bis-EA) increased the apparent molecular mass determined with nonreducing SDS/PAGE from approximately 85 to approximately 195 kDa. After cross-linking, but not before, coexpressed, differentially epitope-tagged DAT molecules, solubilized in Triton X-100, were coimmunoprecipitated. Thus, the 195-kDa complex was a homodimer. Cross-linking of DAT did not affect tyramine uptake. Replacement of Cys-306 with Ala prevented cross-linking. Replacement of all of the non-disulfide-bonded cysteines in the extracellular and membrane domains, except for Cys-306, did not prevent cross-linking. We conclude that the cross-link is between Cys-306 at the extracellular end of TM6 in each of the two DATs. The motif GVXXGVXXA occurs at the intracellular end of TM6 in DAT and is found in a number of other neurotransmitter transporters. This sequence was originally found at the dimerization interface in glycophorin A, and it promotes dimerization in model systems. Mutation of either glycine disrupted DAT expression and function. The intracellular end of TM6, like the extracellular end, is likely to be part of the dimerization interface.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Evidence that polymorphonuclear neutrophils infiltrate into the developing corpus luteum and promote angiogenesis with interleukin-8 in the cow. 21651784

    After ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL.Polymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs.PMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1044
    Nombre del producto:
    Anti-Interleukin-8 Antibody, clone 8M6

  • Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes. 15337822

    Direct stretch of beta1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl(-) SAC resemble those of the volume-sensitive Cl(-) current, I(Cl,swell). Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for beta1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 microM), an AT1R competitive antagonist, blocked Cl(-) SAC but did not significantly alter the background Cl(-) current in the absence of integrin stretch. AT1R signaling is mediated largely by H(2)O(2) produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 microM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl(-) SAC elicited by stretch and the background Cl(-) current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl(-) SAC as well as a large fraction of the background Cl(-) current. With continuing integrin stretch, Cl(-) SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl(-) current that was rapidly and completely blocked by DPI (60 microM). Moreover, exogenous H(2)O(2) (10, 100, and 500 microM), the eventual product of NADPH oxidase activity, also activated Cl(-) SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H(2)O(2) scavenger, attenuated the response to stretch. Application of H(2)O(2) during NADPH oxidase inhibition by either DPI (60 microM) or AEBSF (0.5 mM) did not fully reactivate Cl(-) SAC, however. These results suggest that stretch of beta1-integrin in cardiac myocytes elicits Cl(-) SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl(-) SAC, providing a second regulatory pathway.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2250
    Nombre del producto:
    Anti-Integrin β1 Antibody, clone 21C8

  • Morphological and neoplastic transformation of C3H/10T1/2 Cl 8 mouse embryo cells by insoluble carcinogenic nickel compounds. 2548861

    We studied induction of cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts by soluble and insoluble carcinogenic nickel compounds. Soluble nickel sulfate and nickel chloride caused dose-dependent cytotoxicity in the concentration range from 0.5 microM to 100 microM after 48 hr treatments, but neither compound induced morphological transformation even at concentrations causing up to 94% cytotoxicity. Insoluble nickel subsulfide, nickel monosulfide, and nickel oxide caused dose-dependent cytotoxicity and a low, dose-dependent frequency of morphological transformation in the concentration ranges from 0.5 to 40 microM, 5 to 50 microM, and 50 to 400 microM, respectively, after 48 hr exposure of cells to these compounds. Foci were predominantly of type II morphology; type III foci were rare. The insoluble nickel compounds studied caused no induction of base substitution mutations to ouabain resistance in 10T1/2 cells over concentration ranges that induced morphological transformation. Nickel subsulfide and nickel monosulfide were taken into cells by phagocytosis, since particles were visible in intracytoplasmic vacuoles. Numerous nickel oxide particles were found associated with cells, but true phagocytic uptake was difficult to detect since no vacuoles were observed. We twice cloned type II and type III foci induced by insoluble nickel compounds, established independent cell lines, and characterized their phenotypes. Four of seven of these cell lines had three- to fourfold increased saturation densities compared to 10T1/2 cells, formed type II and type III foci in reconstruction assays, and grew in soft agarose. One cell line induced by nickel oxide formed tumors in nude mice. These data indicate that insoluble carcinogenic nickel compounds induced type II foci in 10T1/2 cells, some of which were tumorigenic, and that the 10T1/2 cell system is suitable for studying mechanisms of nickel compound-induced morphological transformation in mammalian cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    5100
    Nombre del producto: